Title

Author

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Dr. David Hill

Abstract

Islet transplants have been successfully used as treatment for diabetes, but are limited by shortages of cadaveric insulin-producing β-cells. An alternate source may be the dedifferentiation, expansion, and subsequent redifferentiation of pancreatic islets or β-cells using in vitro techniques prior to transplant. Based on protocols which described the dedifferentiation of human islets to ductal-like cells, we hypothesized that neonatal mouse islets could be similarly dedifferentiated in vitro. Dedifferentiation techniques produced significant duct-like cells, but redifferentiation to insulin-expressing cells was limited. RIPCre;Z/AP+/+ mice were consequently utilized to lineage trace β-cell fate during culture by a human placental alkaline phosphatase (HPAP) reporter. The proportion of HPAP+ β-cells decreased significantly in culture, but the rare remaining cells expressed the ductal marker CK19. Flow cytometric sorting of β-cell subpopulations from whole pancreas showed that the HPAP+CK19+ cells had derived from insulin-positive, glucose-transporter-2-negative (Ins+Glut2-) cells, previously purported as insulin-expressing progenitor cells. In P7 mouse pancreas, these Ins+Glut2- cells represent 3.5% of all insulin+ cells, the majority of which were found outside of islets within β-cell aggregates (BCA, <5 β-cells). Ins+Glut2- cells demonstrated greater proliferation rates in vivo and in vitro as compared to Ins+Glut2+ cells, and a subset could differentiate into endocrine, ductal, and neural lineages. We sought to quantify the presence of these progenitor cells throughout life in both mouse and human pancreata, and how their abundance and location changes with age. The presence of Ins+Glut2- cells in human and mouse pancreas demonstrated similar distributions and iii ontogenies, being more abundant in BCA than islets at all ages sampled, and decreasing with age. Finally, neonatal rodents can regenerate β-cell mass after streptozotocin (STZ) exposure, but this response is mitigated in adulthood. As STZ accesses the β-cell via Glut2, we hypothesized that the β-cell regenerative capacity in early life involves mobilization of Ins+Glut2- cells, and used RIPCreER;Z/AP+/+ mice to trace these during damage and regeneration. We found that Ins+Glut2- cells indeed survived STZ exposure, and subsequently matured into Ins+Glut2+ cells. These Ins+Glut2- cells represent a transitional cell type, the majority of which contribute to pancreas maturation, and a subset of which retains multipotential lineage capability.